In vitro metabolic conversion of aflatoxins and benzo(alpha)pyrene to nucleic acid-binding metabolites.
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An aflatoxin B1 metabolite was found to become covalently bound to rat liver RNA and calf thymus DNA in vitro, and it formed complexes with increased spectral absorbance in the 360 nm region. The formation of such complexes was reduced nicotinamide adenine dinucleotide phosphate and microsome dependent, was inhibited by theta-diethylaminoethyl diphenylpropylacetate-HC1, and by CO and N2, when the latter were used to replace the gas phase of the incubations. The formation of the complexes was enhanced about 2-fold with cicrosomes from phenobarbital-treated rats but not from 3-methylcholanthrene-treated rats. More binding was observed with DNA than RNA. Dentured DNA was about 70% as effective as native DNA. Nucleic acids from various sources showed the following order of binding potency: DNA from Micrococcus luteus greater than DNA from calf thymus equal to DNA from rat liver greater than RNA from rat liver greater than transfer RNA from rat liver. In the presence of reduced nicotinamide adenine dinucleotide phosphate and microsomes from phenobarbital-treated rats, aflatoxin G1 was also converted into metabolite(s) that became covalently bound to nucleic acids and formed complexes with increased spectral absorbances in the 360 nm region: this reaction was also inhibited by theta-diethylaminoethyl diphenylpropylacetate-HC1. Under the same conditions, aflatoxin B2, aflatoxin G2, aflatoxin B2a, and "Compound 11," which lack a C2-C3 double bond, did not show any noticeable binding to either DNA or RNA. These data strongly support the concept that the microsomal mixed-funciton oxygenase-catalyzed oxidation of the C2-C3 double bond of aflatoxins is a prerequisite for the formation of nucleic acid-binding metabolites. Microsomes from untreated, phenobarbital-treated, and 3-methylcholanthrene-treated rats were compared in vitro for their ability to catalyze the formation of DNA-binding metabolites from aflatozin B1 and benzo(a)pyrene. In assays involving benzo(a)pyrene, microsomes from 3-methylcholanthrene-treated rats were 12- and 5-fold more active than microsomes from untreated and phenobar-bital-treated rats, respectively. This is in contrast to the results obtained with aflatoxin B1 and suggests that different enzymes in the hepatic microsomal mixed-function oxygenase complex are involved in the generation of reactive metabolites from various polycyclic hydrocarbons. (+info)
Mapping the alpha-subunit site photolabeled by the noncompetitive inhibitor [3H]quinacrine azide in the active state of the nicotinic acetylcholine receptor.
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We have characterized the time-resolved labeling of a site on the Torpedo californica electrocyte acetylcholine receptor (ACHR) by the photoreactive noncompetitive inhibitor derivative quinacrine azide (QA). The dependence of [3H]QA labeling on acetylcholine (ACH) concentration and on time is consistent with the preferential labeling by [3H]QA of ACHR in the open state. The ACH-dependent [3H]QA labeling, which was associated predominantly with the alpha-subunit, was blocked by other noncompetitive inhibitors including quinacrine, chlorpromazine, proadifen, histrionicotoxin, and bupivacaine. alpha-Subunit from ACHR labeled with [3H]QA 20 ms after the addition of ACH was cleaved with CNBr, and the fragments were separated by high pressure liquid chromatography. A peptide containing a major site of specific labeling was purified on two different reverse-phase columns. By N-terminal sequencing, amino acid composition, binding to mercurial-agarose, and apparent molecular weight, this [3H]QA-labeled peptide was identified as alpha-208-243, a CNBr fragment containing the putative membrane-spanning helix M1. (+info)
Multiple actions of substance P that regulate the functional properties of acetylcholine receptors of clonal rat PC12 cells.
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1. The effects of substance P (SP) on each of the kinetic components of reversible desensitization (measured at 4 degrees C) and also on irreversible deactivation (measured at 22 degrees C) of the nicotinic acetylcholine receptor on PC12 cells were examined by 22Na+ influx measurements of the functional state of the receptor. 2. In the absence of agonists, SP converts the acetylcholine receptors in a time- and concentration-dependent manner, to a state that is not responsive to agonist. Upon removal of the peptide, this effect was reversible and the kinetics of the recovery of the permeability response were analysed to provide further characterization of the non-responsive state. Following exposure of cells to SP (10 microM) for 3 or more min, recovery was by a first-order process (time constant, t1/2 = 2.1 min), the same value, within experimental error, as that observed for recovery measured after the initial rapid phase of agonist-mediated desensitization. 3. In the presence of agonist, SP caused a strong enhancement of both the rate and extent of agonist-mediated desensitization. This effect was observed even at concentrations of peptide which produced only a small extent of desensitization when incubated alone. For 500 microM-carbamylcholine, the equilibrium level of desensitization (approximately 85% loss of the permeability response) was achieved at 4 degrees C in about 20 min by a biphasic process, while in the presence of 1.0 microM-SP, complete (100%) desensitization occurred by a single rapid exponential phase characterized by a t1/2 of 20 s. 4. The concentration of carbamylcholine required to produce half-maximal desensitization at equilibrium, Kdes, was 94 microM and was reduced by 6-fold in the presence of 0.3 microM-SP. 5. A mechanistic model is presented in which the receptor is viewed as existing in a dynamic conformational equilibrium between an activatable state Rc and the initial desensitized state Rd. It is proposed that SP binds preferentially to the Rd state and thus can allosterically (1) stabilize the receptor in the absence of agonist in that state, and (2) enhance, in an even lower concentration range, both the rate and extent of agonist-mediated stabilization of the receptor in the Rd state. 6. The second, slower component of agonist-mediated desensitization is, in contrast, inhibited by SP. This desensitization step appears to involve a covalent modification of the initial desensitized state (Rd) and is dependent on Ca2+. SP may exert this inhibitory effect by limiting the access of Ca2+ to an intracellular site of action.(ABSTRACT TRUNCATED AT 400 WORDS) (+info)
Benzo(a)pyrene metabolism by murine spleen microsomes.
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The immunosuppressive actions of benzo(a)pyrene have been proposed to be mediated by reactive metabolites rather than the parent compound. Reactive metabolites which suppress splenic humoral immune responses are thought to be generated within the spleen rather than in distant tissues. Although the spleen has been shown to be capable of metabolizing benzo(a)pyrene, the relative amounts and types of metabolites generated have not been determined. In this study, high-pressure liquid chromatography was used to separate benzo(a)pyrene metabolites generated by splenic microsomes. The major metabolites generated by the splenic microsomal preparations of untreated female B6C3F1 mice were found to be the 9,10- and 7,8-dihydrodiols and 9-, 7-, and 3-hydroxy benzo(a)pyrene. The 1,3-, 3,6-, and 6,12-diones and 4,5-dihydrodiol constituted only a small fraction of the metabolites generated. The generation of all metabolites were inhibited by alpha-naphthoflavone and antiserum to NADPH-cytochrome P-450 reductase, whereas SKF 525-A had only a minimal effect. Dihydrodiol production was completely inhibited by the epoxide hydrolase inhibitor, trichloropropylene oxide. Benzo(a)pyrene pretreatment of mice produced a dramatic increase in the amount of metabolites formed; however, the pattern of metabolites remained similar to that generated by splenic microsomes of untreated mice. The role of prostaglandin synthetase in generating these metabolites was also examined. The addition of arachidonic acid in place of NADPH resulted in the formation of only quinones. Dihydrodiols and phenols were undetectable. The results of this study indicate that splenocytes may be capable of generating the 7,8-dihydrodiol, the precursor to the highly reactive 7,8-dihydrodiol-9,10-epoxide. Furthermore, the addition of the 7,8-dihydrodiol-9,10-epoxide to splenocyte cultures resulted in a decreased in vitro antibody forming cell response to sheep red blood cells. Thus, benzo(a)pyrene-induced immunosuppression may be mediated by the dihydrodiol-epoxide generated within the spleen. Since benzo(a)pyrene exposure was found to increase its own metabolism, immunosuppression produced by the administration of benzo(a)pyrene over several days may be the result of an increased production of immunosuppressive metabolites. The pattern of metabolites generated and the effects of the two types of cytochrome P-450 inhibitors suggests that the major isozyme of cytochrome P-450 that mediates the metabolism of benzo(a)pyrene within the spleen of untreated mice may be similar to the isozyme induced in the liver upon pretreatment with polycyclic aromatic hydrocarbons. (+info)
Development of desensitization during repetitive end-plate activity and single end-plate currents in frog muscle.
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1. The amplitudes of end-plate currents (EPCs) in short trains of fifteen to seventeen EPCs at 10 Hz were depressed in the presence of 10 microM-proadifen when acetylcholinesterase (AChE) was inhibited. 2. The proadifen-induced EPC depression was voltage-dependent and the effect was more pronounced at negative membrane potentials. 3. In the presence of proadifen, the mean amplitude of miniature end-plate currents (MEPCs) was reduced by 36% 5 s after the EPC train as compared with MEPCs before the train. 4. Without proadifen, but with inhibited AChE, an increase of temperature from 20 to 26 degrees C and elevation of external Ca2+ from 1.8 to 2.5 mM led to EPC amplitude depression in the train, which was also potential-dependent. 5. After AChE inhibition, proadifen (10 microM) progressively shortened MEPC decay without significant reduction of amplitude up to 40 min of exposition. MEPCs were not affected by proadifen when AChE was active. 6. It is concluded that these postsynaptic effects of proadifen can be explained neither by its action on the resting acetylcholine receptors (AChR) nor on open ion channels but are due to its desensitization-promoting action. (+info)
Demonstration of different contractile mechanisms for angiotensin II and des-Asp1-angiotensin II in rabbit aortic strips.
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Evidence of selective inhibition, differences in dose-response relationships, and cross-tachyphylaxis studies suggest that separate receptors and/or mechanisms may be involved in responses to angiotensin (Ang), [Sar1]Ang II, and Ang III (= des-Asp1-Ang II). The extracellular Ca2+ requirement for contractile responses induced by angiotensin peptides and norepinephrine was determined in rabbit aortic strips. Responses to K+ and [Sar1]Ang II were attenuated markedly by treatment with SKF-525A, verapamil, or Ca2+-free buffer. The response to Ang II was not impaired by verapamil, was blocked partially by SKF-525A, and was reduced markedly in Ca2+-free medium. Norepinephrine- and Ang III-induced contractions were not dependent on extracellular Ca2+. K+, Ang II, and [Sar1]Ang II required extracellular Ca2+ to induce contraction of the rabbit aorta. The data indicate that Ang III may have a mechanism of action that differs from that of [Sar1]Ang II and Ang II. (+info)
Degradation of aflatoxin by Aspergillus flavus.
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Aflatoxin degradative activity was demonstrated in 6- to 12-d-old intact mycelium and cell-free extracts of Aspergillus flavus. The addition of cycloheximide, SKF 525-A or metyrapone to cultures of A. flavus prevented subsequent degradation of the aflatoxins, while in cell-free extracts degradation was inhibited by SKF 525-A, metyrapone and cytochrome c but not by KCN. In cell-free extracts, aflatoxin degradation was enhanced by NADPH and NaIO4. The results suggest the involvement of cytochrome P-450 monooxygenases in the aflatoxin degradative activity of A. flavus. (+info)
Inhibitors of the cytochrome P-450 enzymes block the secretagogue-induced release of corticotropin in mouse pituitary tumor cells.
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A mouse pituitary tumor cell line (AtT-20) releases corticotropin (ACTH) in response to a number of secretagogues, including corticotropin-releasing factor (CRF), beta-adrenergic agents, N6,O2'-dibutyryladenosine 3',5'-cyclic monophosphate (Bt2 cAMP), and potassium. The stimulation of ACTH secretion induced by the secretagogues can be blocked by inhibitors of the enzymes that generate (phospholipase A2) and metabolize (lipoxygenase and epoxygenase) arachidonic acid. The phospholipase A2 blockers mepacrine and p-bromophenacylbromide inhibited the ACTH release induced by secretagogues. The lipoxygenase inhibitors nordihydroguaieretic acid, butylated hydroxytoluene, and icosatetraynoic acid abolished the ACTH secretion induced by secretagogues, whereas indomethacin, a cycloxygenase inhibitor, did not. Blockers of the cytochrome P-450 epoxygenase, such as SKF 525A and piperonyl butoxide, compounds that have different molecular structures, also suppressed secretagogue-induced ACTH release. These findings suggest that metabolites of arachidonic acid formed via the epoxygenase and/or the lipoxygenase pathway are involved in the stimulation of ACTH release caused by secretagogues. (+info)